Download Hydrogen bonds in the binding of some polyphenols from Quebracho

Hydrogen bonds in the binding of some polyphenols from Quebracho
(Schinopsis spp.) to soybean meal protein under in vitro ruminal conditions1
C. Velázquez2, M. Suárez2, M. Pabón2 y Juan Carulla3
Universidad Nacional de Colombia. Bogotá, Colombia
ABSTRACT: The extent of binding to soya protein of a commercial tannin (Quebracho), its ethyl acetate extract, the
major putative compound (fisetinidol-(4,8)-cathequin-(6,4)-fisetinidol), and the acetylated and methylated derivatives of
the major latter was evaluated incubating them with soybean meal under in vitro ruminal conditions for 48h. The protein
binding activity was estimated measuring changes in ammonia concentration when the tannin was incubated with soybean
meal at a proportion of 8% of the dry weight. As compared to soybean meal alone (control), ammonia concentration decreased by 19, 27, and 31% for quebracho, the ethyl acetate extract of quebracho and the major compound, respectively, indicating concentration of the protein binding activity. Acetylation or methylation of the major compound resulted in corresponding decreases in the concentration of ammonia by 21 and 6%. This suggested that as the possibility of hydrogen bond
formation decreases (being less with methylation than with acetylation), the binding to the protein is reduced but is not
eliminated completely. Therefore, our hypothesis is that the main interactions between fisetinidol-(4,8)-cathequin(6,4)-fisetinidol and soybean protein in an in vitro ruminal system are hydrogen bonds and that only a few hydrophobic interactions are present.
Key words: Fisetinidol-(4,8)-cathequin-(6,4)-fisetinidol, bypass protein, hydrogen bonds, polyphenols, Schinopsis spp
©2002 ALPA. Todos los derechos reservados
Arch. Latinoam. Prod. Anim. 2002. 10(3): 171-174
Puentes de hidrógeno en la ligación de algunos polifenoles de Quebracho
(Shinopsis spp.) a proteína de soya en un sistema ruminal in vitro
RESUMEN: Se evaluó la capacidad de ligación a proteína de soya de un tanino comercial (quebracho), su extracto en
acetato de etilo, el compuesto causativo mayoritario (fisetinidol-(4,8)-cathequin-(6,4)-fisetinidol), y los derivados acetilado
y metilado de éste, incubándolos con torta de soya en un sistema ruminal in vitro durante 48 h. Esta capacidad se estimó midiendo los cambios en la concentración de amonio al incubar el tanino con la torta de soya en una relación tanino/proteína de
8% (p/p materia seca). Con relación a la torta de soya sola (control), las concentraciones de amonio disminuyeron en 19, 27 y
31% para el quebracho, el extracto de quebracho en acetato de etilo y el compuesto mayoritario, respectivamente, indicando
una concentración de la actividad de ligación a la proteína de soya. La acetilación o la metilación del compuesto mayoritario
resultó en disminuciones correspondientes en la concentración de amonio en 21 y 6%. Esto sugiere que a medida que la posibilidad de formación de puentes de hidrógeno disminuye (menor con metilación que con acetilación), la unión a la proteína
se debilita, pero no se elimina completamente. Por lo tanto, se postula que las principales interacciones entre el fisetinidol-(4,8)-cathequin-(6,4)-fisetinidol y la proteína de soya en un sistema ruminal in vitro son los puentes de hidrógeno y que
soló unas pocas interacciones hidrofóbicas estarían presentes.
Palabras clave: Fisetinidol-(4,8)-cathequin-(6,4)-fisetinidol, puentes de hidrógeno, polifenoles, proteína pasante,
Schinopsis spp
Recibido Octubre 18, 2001. Aceptado Agosto 27, 2002
E-mail: [email protected]
We acknowledge the financial support of Colciencias and Universidad Nacional de Colombia.
Departamento de Química.
Facultad de Medicina Veterinaria y de Zootecnia.
1
2
3
171
172
Velázquez et al.
Introduction
Addition of tannin sources to ruminant diets may improve the efficiency of nitrogen utilisation under certain
conditions. Some studies have shown an increase of amino
acid flow to the duodenum and amino acid absorption when
tannins are included in the diet (Jones and Mangan, 1977;
Egan and Utyatt, 1980; Waghorn et al., 1987), whereas
other experiments have demonstrated that tannins reduce
feed intake, protein and dry matter digestibility and worsen
animal performance (Barry and Duncan, 1984; Reed et al.,
1990). In vitro experiments in our laboratory have shown
that a commercial tannin (Quebracho) decreases ammonia,
isobutyric and isovaleric acid production from soybean
meal, indicating binding of the tannin to soya protein, thus
decreasing its in vitro availability for fermentation (Gonzalez et al., 1998). Hydrogen bonds and hydrophobic interactions have been reported as the main forces in the binding
of tannins to proteins (Siebert et al., 1996). Hydrogen
bridges are formed mainly between carbonyl groups of the
peptide bond and OH groups of phenols, while hydrophobic
interactions occur between the aromatic part of two compounds and involve mainly the pyrrol residue of proline
(Oh et al., 1980)
In the present investigation we isolated the major compound responsible for protein binding under in vitro ruminal conditions from the ethyl acetate extract of a commercial tannin (Quebracho). To assess the effects of hydrogen
bond formation between this compound and soybean meal
protein, the former was acetylated and methylated and these
derivatives were also tested using the same system. Ammonia concentration was used to estimate the extent of binding
of the tannin source and its extract or the major compound
and its derivatives to soya protein. Assuming that ruminal
micro-organisms can not degrade protein that is bound to
tannin, the concentration of ammonia should be inversely
proportional to the extent of binding.
Materials and Methods
Samples. The soybean meal and a semipurified commercial tannin, Quebracho (Shinopsis spp.), produced by
Indunor S. A. (Buenos Aires, Argentina), used were analyzed for contents of moisture (AOAC-7.007, 1984) ash
(AOAC-7.009, 1984) and neutral detergent fiber (Van
Soest et al., 1991). The latter fraction was assayed without
sodium sulfite, alpha amylase or residual ash in the case of
tannins but with addition of amylase for soybean meal. Soybean meal was analysed for crude protein (AOAC-7.00337.037, 1984) and soluble nitrogen according to Van Soest et
al. (1995). Total phenol and condensed tannins of the semipurified commercial product were determined using the
Prussian Blue method (Price and Buttler, 1977) and butanol/HCl method (Porter et al., 1986), respectively. The
dried ethyl acetate extract of Quebracho, the major com-
pound, and acetylated and methylated derivatives of the latter were also tested.
Isolation and characterisation of compound C. An
aqueous solution of the commercial tannin (500 g/L) was
succesively extracted liquid-liquid with petroleum ether,
dicloromethane, and finally ethyl acetate. The ethyl acetate
phase was concentrated to dryness (39.9 g) and separated
by column chromatography on silica gel, eluting with dicloromethane, dicloromethane: ethyl acetate in two proportions (1:1 and 1:4), and ethyl acetate. The ethyl acetate fraction was concentrated to dryness (19 g). A part of this fraction (5 g) was chromatographed on silica gel, eluted with dicloromethane: ethanol (continuous gradient). The fractions
eluted with ethanol contained mainly compound C. This
fraction was purified by repeated column chromatography
on silica gel, obtaining 0.7 g of pure C. This compound was
acetylated (Ac2O/pyridine) and methylated (diazomethane)
to yield C1 and C2, respectively. The structure of the isolated compound and its derivatives were elucidated by
spectroscopic methods comparing the structure of the methylated derivative with similar compounds (Botha et al.,
1982; Viviers et al., 1983).
Soybean and tannin mixtures. Soybean meal (100 mg)
was weighed in 100 mL plastic tubes and 8 mg of the commercial tannin, the dried ethyl acetate extract, C, C1 or C2
dissolved in McDougall buffer (5 mL) was added. Triplicate
tubes were used for each tannin and for a control (soybean
meal alone). Tubes were placed in a thermostat at 39°C for
12h to allow tannin and protein to interact (Figure 1).
Ruminal in vitro incubation. In this work only the first
part (degradation by rumen bacteria) of the Tilley and Terry
(1963) procedure was used. Ruminal fluid was collected
from two steers grazing a pasture of kikuyo grass (Pennisetum clandestinum) and white clover (Trifolium repens), filtered through cheesecloth and kept at 39°C under CO2 for
30 minutes. Thereafter, 50 mL of diluted ruminal fluid (ruminal fluid:McDougall buffer-without urea 1:4) were
added to tubes that contained soybean meal alone (control)
or tannin-treated soybean meal. After gasifying with CO2,
tubes were sealed with rubber stoppers provided with bunsen valves, and incubated at 39°C for 48 h, shaking periodically. Ammonia concentration was determined in the fluid
from the incubation tubes.
Ammonia determinations. Aliquots of 4 mL were
taken from each ruminal incubation tube and acidified with
80 mL of H2SO4 (80% v/v). Aliquots of 1mL were centrifuged at 9000 xg for 10 min. Ammonia concentration was
determined in the supernatant using a colorimetric method
(McCullough, 1967).
Results
The composition of soybean meal and quebracho are
presented in Table 1.
The major compound C, isolated from the ethyl acetate
extract of a commercial sample of quebracho (Schinopsis
Tannin binding to soya protein
OR
OR
O
RO
OR
OR
OH
RO
O
RO
RO
RO
OH
RO
C R=H
C1 R=Ac
C2 R=Me
Fig 1. Chemical structures of compounds C, C1 and C2.
Table 1.
Composition of soybean meal and tannin
sources.
Soybean meal
Quebracho
DM (%)
87.8
91.4
CP (%)
50.2
23.0
Soluble protein (%)
NDF (%)
12.3
Total phenols (%)
39.3
0.82
Condensed tannins (A × mg )
0
Hydrolysable tannins (%)
DM=DryMatter,CP=CrudeProtein,NDF=NeutralDetergentFiber.
% of CP.
As gallic acid.
1
-1
550
2
1.
2.
Table 2.
Ammonia concentration after addition of
quebracho, the ethyl acetate extract or the major
compound to soybean meal in an in vitro
ruminal incubation system
Change in ammonia
Ammonia
concentration concentration relative
to control (%)
(NH mg × dL )
Soybean meal (Control)
20.5 ± 0.4
Quebracho
16.7 ± 0.3
-19
Ethyl acetate extract
15.0 ± 0.2
-27
Major compound (C)
14.2 ± 0.2
-31
Acetylated compound (C1)
16.2 ± 0.3
-21
Methylated compound (C2)
19.3 ± 0.4
-6
Treatment
3
spp.), was characterised as fisetinidol-(4,8)-cathequin-(6,4)
-fisetinidol by the spectroscopic methods used.
Upon testing the in vitro system, lower levels of ammonia were obtained relative to the control when quebracho,
the ethyl acetate extract, or the major compound C were
added to soybean meal at the 8% level. Higher levels of ammonia were found with the acetylated (C1) or methylated
(C2) derivations than when C itself was added (Table 2).
Discussion
OH
O
173
-1
In the present investigation an in vitro ruminal system
using a 48h incubation time was used. Other researchers
have suggested that to estimate ruminal protein degradation
shorter incubation times and an inhibitor of both deamination and ammonia uptake should be used (Broderick, 1978;
Broderick, 1987). Our system did not take into account the
limitations of interpretation associated with microbial synthesis and incorporation of ammonia to the bacteria at
longer incubation times. However, the results showed that
the system is sensible to the effects of adding different types
of compounds. This system may not be accurate to measure
the absolute extent of degradation but it could be used to
compare the effects of different types of compounds on protein degradability.
The present results agree with those of experiments in
vivo and in vitro showing a decrease in ruminal protein
degradation by addition of tannins (Zelter and Leroy,
1966; Driedger and Hatfield, 1972; Barry and Manley,
1984; Asquith and Butler, 1986; Waghorn et al., 1987). In
our experiment, effect on ammonia levels decreased in the
following order: Quebracho < Ethyl acetate extract < C,
indicating concentration of the protein binding capacity.
Twice as much protection of soybean meal from ruminal
degradation
was
found
with
C
(fisetinidol-(4,8)-cathequin-(6,4)-fisetinidol) than with the commercial quebracho. According to the molecular structure
of compound C interactions with soybean meal protein
might be expected through hydrogen bridges or hydrophobic interactions. In order to evaluate the contribution of
these two types of bonding, the acetylated and methylated
derivatives of compound C were synthesised and tested in
the same system. Acetylation or methylation of compound
C decreased ammonia levels, relative to the control by 21
and 6%, respectively, indicating that there is still some
binding to protein in both compounds. In the acetylated
compound the carbonyl group could interact with protein
to form hydrogen bonds while this is not so in the methylated derivative. However, hydrophobic interactions could
still be present between the aromatic rings of polyphenol
and protein, explaining the remaining activity of the methylated compound. Similar conclusions have been reached
upon lowering the pH in a system where tannin and protein
are present. Hydrogen bonds are susceptible to pH
changes and decreasing pH reduces but does not eliminate
the binding of tannin and protein, which suggests the pres-
174
Velázquez et al.
ence of both hydrogen bonds and hydrophobic interactions
(Oh et al.,1980; Fajardo et al., 1998; Gonzalez et al., 1998).
Conclusion
The main interactions between the major putative compound C obtained from quebracho and soybean meal protein are probably hydrogen bonds but hydrophobic interactions also contribute to this binding.
Literature Cited
AOAC. 1984. Association of Official Analytical Chemists. Washinton, D.
C.
Asquith, T. N. and L. G. Butler. 1986. Interactions of condensed tannins
with selected proteins, Phytochemistry 25:1591.
Barry, T. N. and S. J. Duncan. 1984. The role of condensed tannins in the
nutrition value of
for sheep. 1. Voluntary intake.
Br. J. Nutr. 51:485.
Barry, T. N.and T. R. Manley. 1984. The role of condensed tannins in the
nutrition value of
for sheep. 2. Quantitative digestion of carbohydrates and proteins. Br. J. Nutr. 51:493.
Botha, J. J., P. M. Viviers, D. A. Young, C. du Preez, D. Ferreira, and D.
G., Roux. 1982. Synthesis of condensed tannins. Part 5. The first angular [4,6: 4,8]-triflavonoids and their natural counterparts. J. Chem
Soc. Perkin Trans. 1:527.
Broderick, G. A., 1978.
procedures for estimating rates of ruminal
protein degradation and proportions of protein escaping the rumen
undegraded. J. Nutr. 108:181.
Broderick, G. A., 1987. Determination of protein degradation rates using a
rumen
system containing inhibitors of microbial nitrogen
metabolism. J. Dairy Sci. 58:463.
Driedger, A. R., and E. E. Hatfield. 1972. Influence of tannins on the nutritional value of soybean meal for ruminants. J. Anim. Sci. 34:465.
Lotus pedunculatus
Lotus pedunculatus
In vitro
in vitro
Egan, A. R. and M. J. Ulyatt. 1980. Quantitativedigestion of fresh herbage
by sheep. VI. Utilization of nitrogen in five herbages. J. Agric. Sci.
Camb. 9:45.
Fajardo, C. H., M. L. Pabón y J. Carulla. 1998. Estudio de la interacción
entre proteína de soya y taninos. XI Congreso Colombiano de
Química y 1ª Feria Internacional de Química Mem.
González,S., J. Carulla,and M. L. Pabón.1998. Effectsof tanninson in vitro ruminal protein and dry matter degradation of soybean meal and
ryegrass. J. Dairy Sci. 69 (Suppl. 1):81.
Jones, W. T. and J. L. Mangan. 1977. Complexes of the condensed tannins
of sainfoin (
.) with fraction 1 leaf protein
and with submaxillary mucoprotein, and their reversal by polyethylene glycol and pH. J. Sci. Food Agric. 28:126.
McCullough, H. 1967. The determination of ammonia in whole blood by
direct colorimetric method, Clin. Chem. Acta 17:297.
Oh, H., J. Hoff, G. Armstrong, and L. Haff. 1980. Hydrophobic interactions in tannin-protein complexes. J. Agric. Food Chem. 28:394.
Reed, J. D., H. Soller, and A. Woodward.1990. Foddertree and straw diets
for sheep:intake,growth,digestibilityand the effectsof phenolicson
nitrogen utilization. Anim. Feed Sci. Tech. 30:39.
Siebert, K. J., N. V. Troukhanova, and P. Y. Lynn. 1996. Nature of
polyphenol-protein interactions. J. Agric. Food Chem. 44:80.
Tilley, J. M. A. and R. A. Terry. 1963. Two stage technique for the
digestion. Br. Grass. Soc. 18:104.
Viviers,P. M., H. Kolodziej,D. A. Young, D. Ferreira,and D. Roux. 1983.
Synthesis of condensed tannins. Part II. Intramolecular enantiomerism of the constituent units of tannins from the Anacardiaceae: Stoicheiometric control in direct synthesis: derivation of 1H nuclear
magnetic resonance parameters applicable to higher oligomers. J.
Chem. Soc. Perkin Trans. 1:2555.
Waghorn, G. C., A. Jhon, and W. T. Jones. 1987. Nutritive value of Lotus
corniculatus containing low and medium concentrations of condensed tannins for sheep. Proc. N. Z. Soc. Anim. Prod. 47:25.
Zelter, S. Z. and F. Leroy. 1966. Schutz der Nahrungsproteine gegen microbielle Desaminierung im Pansen. Z. Tierphysiol. Tierernahr. Futtermittelkde 22:3.
in
vitro
Onobrychis vicifolia Scop
in vitro